[Structure of ion transport ATPase--progress in active site structure and primary structure of Na+, K+-ATPase].
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Amino Acid Sequence↗
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[Stereochemical theory of the 3-dimensional structure of globular proteins. II. Transition from the highly helical intermediate structure to the native structure].
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Models, Chemical↗
At nonzero temperatures, stacked structures of methylated nucleic acid base pairs and microhydrated nonmethylated nucleic acid base pairs are favored over planar hydrogen-bonded structures: a molecular dynamics simulations study.
The dynamic structure of all ten possible nucleic acid (NA) base pairs and methylated NA base pairs hydrated by a small number of water molecules (from 1 to 16) was determined by using molecular dynamics simulations in the NVE microcanonical and NVT canonical ensembles with the Cornell force field (W. D. Cornell, P. Cieplak, C. I. Bayly, I. R. Gould, K. M. Merz, D. M. Ferguson, D. C. Spellmeyer, T. Fox, J. E. Caldwell, P. Kollman, J. Am. Chem. Soc. 1995, 117, 5179). The presence of one water molecule does not affect the structure of any hydrogen-bonded (H-bonded) nonmethylated base pair. An equal population of H-bonded and stacked structures of adenine...adenine, adenine...guanine and adenine... thymine pairs is reached if as few as two water molecules are present, while obtaining equal populations of these structures in the case of adenine...cytosine, cytosine...thymine, guanine... guanine and guanine...thymine required the presence of four water molecules, and in the case of guanine...cytosine, six. A comparable population of planar, H-bonded and stacked structures for cytosine...cytosine and thymine... thymine base pairs was only obtained if at least eight water molecules hydrated a pair. Methylation of bases changed the situation dramatically and stacked structures were favoured over H-bonded ones even in the absence of water molecules in most cases. Only in the case of methyl cytosine...methyl cytosine, methyl guanine...methyl guanine and methyl guanine...methyl cytosine pairs were two, two or six water molecules, respectively, needed in order to obtain a comparable population of planar, H-bonded and stacked structures. We believe that these results give clear evidence that the preferred stacked structure of NA base pairs in the microhydrated environment, and also apparently in a regular solvent, is due to the hydrophilic interaction of a small number of water molecules. In the case of methylated bases, it is also due to the fact that the hydrogen atoms most suitable for the formation of H-bonds have been replaced by a methyl group. A preferred stacked structure is, thus, not due to a hydrophobic interaction between a large bulk of water molecules and the base pair, as believed.
Base Pairing↗
Predicting RNA structures: the model of the RNA element binding Rev meets the NMR structure.
BACKGROUND: How accurate are the predictions of RNA three-dimensional structures? Assessing this accuracy requires the detailed comparison of the prediction with the experimentally determined structure. Previously, sequence variation in RNA aptamers that bind the Rev protein was used to infer a three-dimensional model of the Rev-binding element (RBE) RNA. Although much of this model has been substantiated by subsequent experimental data, its validity remains to be determined by confronting it with the structure determined by NMR spectroscopy. RESULTS: A series of different criteria such as geometric parameters (root mean square deviation, interproton distances, torsions and puckering), helicoidal parameters (base pairing and base stacking) and stability considerations (conformational energies) have been evaluated to identify common and distinguishing structural characteristics of the model and the NMR structure. CONCLUSIONS: The detailed comparison of the two structures reveals striking structural similarities at both the global and local level that validate the RNA modeling approach that we have used. Analysis of the structural differences and the precision of the model suggest that the limitations of the method are related to the amount of structural information available for modeling.
Binding Sites↗
Formation of metastable RNA structures by sequential folding during transcription: time-resolved structural analysis of potato spindle tuber viroid (-)-stranded RNA by temperature-gradient gel electrophoresis.
A model of functional elements critical for replication and infectivity of the potato spindle tuber viroid (PSTVd) was proposed earlier: a thermodynamically metastable structure containing a specific hairpin (HP II) in the (-)-strand replication intermediate is essential for template activity during (+)-strand synthesis. We present here a detailed kinetic analysis on how PSTVd (-)-strands fold during synthesis by sequential folding into a variety of metastable structures that rearrange only slowly into the structure distribution of the thermodynamic equilibrium. Synthesis of PSTVd (-)-strands was performed by T7-RNA-polymerase; the rate of synthesis was varied by altering the concentration of nucleoside triphosphates to mimic the in vivo synthesis rate of DNA-dependent RNA polymerase II. With dependence on rate and duration of the synthesis, the structure distributions were analyzed by temperature-gradient gel electrophoresis (TGGE). Metastable structures are generated preferentially at low transcription rates--similar to in vivo rates--or at short transcription times at higher rates. Higher transcription rates or longer transcription times lead to metastable structures in low or undetectable amounts. Instead different structures do gradually appear having a more rod-like shape and higher thermodynamic stability, and the thermodynamically optimal rod-like structure dominates finally. It is concluded that viroids are able to use metastable as well as stable structures for their biological functions.
DNA-Directed RNA Polymerases↗
Hydrogen bonding and solvent structure in an antigen-antibody interface. Crystal structures and thermodynamic characterization of three Fv mutants complexed with lysozyme.
Using site-directed mutagenesis, X-ray crystallography, and titration calorimetry, we have examined the structural and thermodynamic consequences of removing specific hydrogen bonds in an antigen-antibody interface. Crystal structures of three antibody FvD1.3 mutants, VLTyr50Ser (VLY50S), VHTyr32Ala (VHY32A), and VHTyr101Phe (VHY101F), bound to hen egg white lysozyme (HEL) have been determined at resolutions ranging from 1.85 to 2.10 A. In the wild-type (WT) FvD1.3-HEL complex, the hydroxyl groups of VLTyr50, VHTyr32, and VHTyr101 each form at least one hydrogen bond with the lysozyme antigen. Thermodynamic parameters for antibody-antigen association have been measured using isothermal titration calorimetry, giving equilibrium binding constants Kb (M-1) of 2.6 x 10(7) (VLY50S), 7.0 x 10(7) (VHY32A), and 4.0 x 10(6) (VHY101F). For the WT complex, Kb is 2.7 x 10(8) M-1; thus, the affinities of the mutant Fv fragments for HEL are 10-, 4-, and 70-fold lower than that of the original antibody, respectively. In all three cases entropy compensation results in an affinity loss that would otherwise be larger. Comparison of the three mutant crystal structures with the WT structure demonstrates that the removal of direct antigen-antibody hydrogen bonds results in minimal shifts in the positions of the remaining protein atoms. These observations show that this complex is considerably tolerant, both structurally and thermodynamically, to the truncation of antibody side chains that form hydrogen bonds with the antigen. Alterations in interface solvent structure for two of the mutant complexes (VLY50S and VHY32A) appear to compensate for the unfavorable enthalpy changes when protein-protein interactions are removed. These changes in solvent structure, along with the increased mobility of side chains near the mutation site, probably contribute to the observed entropy compensation. For the VHY101F complex, the nature of the large entropy compensation is not evident from a structural comparison of the WT and mutant complexes. Differences in the local structure and dynamics of the uncomplexed Fv molecules may account for the entropic discrepancy in this case.
Antigen-Antibody Complex↗
Structure generation by reduction: a new strategy for computer-assisted structure elucidation.
A problem common to computer programs for structure elucidation is the efficient and prospective use of the input information to constrain the structure generation process. The input may consist of potentially overlapping substructure requirements and alternative substructure interpretations of spectral data. Other useful information may be structural features that must not be present in the output structures. All of these may interact in a complex manner that is impossible to determine by use of a bond-by-bond structure assembly algorithm. A new method is described called structure reduction. In contrast to structure assembly, this method begins with a set of all bonds and removes inconsistent bonds as structure generation progresses. This results in a more efficient use of the input information and the ability to use potentially overlapping required substructures. Several examples illustrate the application of our computer program COCOA, which uses this method to solve real-world structure elucidation problems.
Chemical Phenomena↗
Structure and bonding of Sr3In11: how size and electronic effects determine structural stability of polar intermetallic compounds.
The binary compound Sr(3)In(11) (SrIn(3.667)) was synthesized and structurally characterized by X-ray diffraction experiments. It crystallizes in the orthorhombic La(3)Al(11) structure type (space group Immm, Z = 2; a = 4.9257(6), b = 14.247(2), c = 11.212(2) A). The crystal structure of Sr(3)In(11) bears features of the monoclinic EuIn(4) structure, which is adopted by SrIn(4), and the prominent tetragonal BaAl(4) structure. Sr(3)In(11) is stable until 550 degrees C. At higher temperatures it decomposes peritectically into SrIn(2) and In. Structural stability and bonding properties of Sr(3)In(11) were investigated by first principles calculations and compared to SrIn(4) in the monoclinic EuIn(4) and the tetragonal BaAl(4) structure. All three structures consist of a three-dimensional, polyanionic, network formed by In atoms and Sr cations encapsulated in cages. For the BaAl(4)-type SrIn(4), In-In network bonding is perfectly optimized. In contrast, the networks of EuIn(4)-type SrIn(4) and Sr(3)In(11) appear hypo- and hyperelectronic, respectively. The formation of Sr(3)In(11) with a composition close to 1:4 and the nonexistence of BaAl(4)-type SrIn(4) is explained by a delicate interplay of size and electronic factors governing structural stability in the In-rich part of the Sr-In system.
Journal Article↗
The Four-Connected Net in the CeCu(2) Structure and Its Ternary Derivatives. Its Electronic and Structural Properties.
The crystallochemistry of and the bonding in the orthorhombic four-connected nets of BaIn(2) (CeCu(2) structure) and of CaPtSn (TiNiSi structure, a derivative of the CeCu(2) structure) are analyzed with approximate molecular orbital calculations. Following the Zintl concept, in BaIn(2) the In(-) ions are isoelectronic with group IV tin and should adopt a four-connected structure. In contrast to alpha-tin, which has a cubic diamond structure, the indium ions in BaIn(2) build up an orthorhombic three-dimensional four-connected net containing distorted tetrahedra and ladder polymers of four-membered rings. In the CeCu(2) structure (space group Imma) two bond angles in these distorted tetrahedra are fixed at 90 degrees. The four-connected net in the CeCu(2) structure is topologically related to the layers in black phosphorus (space group Cmca). In CaPtSn (TiNiSi structure) the orthorhombic four-connected net is formed by (PtSn)(2)(-) ions in an ordered arrangement. Calculations on BaIn(2) and CaPtSn show that the four-connected nets are increasingly stabilized as the valence electron count is increased from 16 to 30 valence electrons per 4 formula units. For more than 30e, the nets are destabilized due to filling of M-E antibonding states. Structural data obtained by precise single crystal investigations for the TiNiSi series CaPdIn (20e), CaPdSn (24e), CaPdSb (28e), and CaAgSb (32e), confirm the results of the extended Hückel calculations. We find an interesting and understandable angular asymmetry of the tetrahedral sites in these ternary compounds.
Journal Article↗
The three-dimensional structure of Asp189Ser trypsin provides evidence for an inherent structural plasticity of the protease.
Trypsin mutant Asp189Ser, first described by Gráf et al. [Gráf, L., Jancsó, A., Szilágyi, L., Hegyi, G., Pintér, K., Náray-Szabó, G., Hepp, J., Medzihradszky, K. & Rutter, W.J. (1988) Proc. Natl Acad. Sci. USA 85, 4961-4965] has played an important role in recent studies on the structural basis of substrate-specific catalysis by serine proteases. The present work reports the three-dimensional structure of this mutant crystallized in unliganded form: the first unliganded rat trypsin structure reported. The X-ray structure of the Asp189Ser trypsin mutant in complex with bovine pancreatic trypsin inhibitor is already known. The X-ray structure of free Asp189Ser rat trypsin revealed that the single amino acid mutation at the bottom of the substrate binding pocket of trypsin resulted in extensive structural changes around the mutated site and in dimerization of the mutant, in contrast with the complexed enzyme the structure of which is practically the same as that of wild-type trypsin. The structural rearrangement in the mutant was shown to be restricted to the activation domain region providing further evidence for the allosteric property of this structural-functional unit of the enzyme. This study supports our view that the plasticity of the activation domain may play an important role in the mechanism of substrate-specific serine protease action.
Allosteric Site↗
On the structural changes of native human alpha2-macroglobulin upon proteinase entrapment. Three-dimensional structure of the half-transformed molecule.
The reconstructions of an intermediate form of human alpha2-macroglobulin (half-transformed alpha2M) in which two of its four bait regions and thiol ester sites were cleaved by chymotrypsin bound to Sepharose were obtained by three-dimensional electron microscopy from stain and frozen-hydrated specimens. The structures show excellent agreement and reveal a structure with approximate dimensions of 195 (length) x 135 (width) and 130 A (depth) with an internal funnel-shaped cavity. The structure shows that a chisel-shaped body is connected to a broad base at the opposing end by four stands. Four approximately 45 A diameter large openings in the body of the structure result in a central cavity that is more accessible to the proteinase than those associated with the native or fully transformed structures. The dissimilarity in the shapes between the two ends of alpha2M half-transformed and the similarity between its chisel-shaped body and that of native alpha2M indicate that the chymotrypsin has cleaved both bait regions in the bottom-half of the structure. Consequently, its functional division lies on the minor axis. The structural organization is in accord with biochemical studies, which show that the half-transformed alpha2M migrates on native polyacrylamide gels at a rate intermediate to the native and fully transformed alpha2M and is capable of trapping 1 mol of proteinase. Even though its upper portion is similar to the native molecule, significant differences in their shapes are apparent and these differences may be related to its slower reaction with a proteinase than the native structure. These structural comparisons further support the view that the transformation of alpha2M involves an untwisting of its strands with an opening of the cavity for entrance of the proteinase and a retwisting of the strands around the proteinase resulting in its encapsulation.
Binding Sites↗
Molecular-mechanics modelling of drug-DNA structures; the effects of differing dielectric treatment on helix parameters and comparison with a fully solvated structural model.
This study analyses the influence that the nature of the dielectric constant has on the final structures obtained from in vacuo molecular mechanics calculations on a drug-DNA complex and compares these structures with the energy minimised complex including explicit solvent molecules. Minimisations have been performed on a proflavine-decanucleotide structure, where the drug was intercalated at the d(CpG) site of the d(GpApTpApCpGpApTpApC) decamer duplex, using two expressions for the dielectric constant: a distance-independent, epsilon ij = EPS, and a distance-dependent, epsilon ij = EPS*Rij, form and for values of EPS from 1 to 8. Significantly different structures are obtained for the distance-independent and the distance-dependent expressions of the dielectric constant. The use of a distance-independent dielectric constant leads to distorted structures, which are very sensitive to slight changes in the value of EPS. The use of a distance-dependent dielectric constant leads to less distorted and more stable structures. The effects on helical parameters are analysed in detail. The structures obtained for different values of EPS (within the distance-dependent formalism) seem to converge for values of EPS equal to 4 or greater. Based on these results a distance-dependent form of the dielectric with an EPS value of 4 is recommended in order to produce reliable refined nucleic acid structures by molecular mechanics. These conclusions have been supported by molecular-mechanics minimisation of the same structure with the inclusion of explicit water molecules and counter-ions.
Base Composition↗
The FSSP database: fold classification based on structure-structure alignment of proteins.
The FSSP database presents a continuously updated classification of 3-D protein folds based on an all-against-all comparison of structures currently in the Protein Data Bank (PDB) [Bernstein et al. (1977) J. Mol. Biol., 112, 535- 542]. The database currently contains an extended structural family for each of 600 representative protein chains which have <25% mutual sequence identity. The results of the exhaustive pairwise structure comparisons are reported in the form of a fold tree generated by hierarchical clustering and as a series of structurally representative sets of folds at varying levels of uniqueness. For each query structure from the representative set, there is a database entry containing structure-structure alignments with its structural neighbours in the representative set and its sequence homologs in the PDB. All alignments are based purely on the 3-D co-ordinates of the proteins and are derived by an automatic structure comparison program (Dali). The FSSP database is accessible electronically on the World Wide Web and by anonymous ftp.
Amino Acid Sequence↗
The structure of orange HgI2. II. Diamond-type structure and twinning.
The metastable orange crystals of HgI(2) comprise three different crystal structures all of which are built from corner-linked Hg(4)I(10) supertetrahedra. Two of the structures are end members with the maximum degree of order (MDO) of a polytypic layer structure. In this paper, the third structure (D) determined from X-ray diffraction, a crystal chemical discussion of the four known tetrahedral HgI(2) structures, and a twinning model are presented. All the various diffraction results published during the past 70 years are now explained. The Hg(4)I(10) supertetrahedra of the tetragonal structure D are corner-linked into two interpenetrating diamond-type networks. The stable red form and the three orange structures show the same cubic densest packing of I atoms and differ only in the distribution of Hg atoms in the tetrahedral voids. Transformations between the structures may involve only movements of Hg atoms, as implied by larger thermal displacement parameters of Hg than of I. A multiply twinned conglomerate of MDO1, MDO2 and D, each structure occurring in three orientations, results in metrically cubic crystals whose Bragg reflections are very close to reciprocal lattice points.
Journal Article↗
Structure and function of the N-linked glycans of HBP/CAP37/azurocidin: crystal structure determination and biological characterization of nonglycosylated HBP.
The three N-glycosylation sites of human heparin binding protein (HBP) have been mutated to produce a nonglycosylated HBP (ng-HBP) mutant. ng-HBP has been crystallized and tested for biological activity. Complete X-ray data have been collected to 2.1 A resolution, and the structure has been fully refined to an R-factor of 18.4% (R(free) 27.7%). The ng-HBP structure reveals that neither the secondary nor tertiary structure have changed due to the removal of the glycosylation, as compared to the previously determined glycosylated HBP structure. Although the primary events in N-linked glycosylation occurs concomitant with polypeptide synthesis and therefore possesses the ability to influence early events in protein folding, we see no evidence of glycosylation influencing the structure of the protein. The root-mean-square deviation between the superimposed structures was 0.24 A (on C alpha atoms), and only minor local structural differences are observed. Also, the overall stability of the protein seems to be unaffected by glycosylation, as judged by the B-factors derived from the two X-ray structures. The flexibility of a glycan site may be determined by the local polypeptide sequence and structure rather than the glycan itself. The biological in vitro activity assay data show that ng-HBP, contrary to glycosylated HBP, mediates only a very limited stimulation of the lipopolysaccharide induced cytokine release from human monocytes. In animal models of fecal peritonitis, glycosylated HBP treatment rescues mice from and an otherwise lethal injury. It appears that ng-HBP have significant effect on survival, and it can be concluded that ng-HBP can stimulate the host defence machinery albeit to a lesser extent than glycosylated HBP.
Animals↗
Smoothing of pixelated finite element models of cancellous bone structures and the effect on the predicted structural properties of the bone.
Many areas of biomedical engineering involve the modelling of biological systems, often using data from medical scanning techniques such as computed microtomography (microCT), and the prediction of the mechanical properties of these systems via finite element models. These models, and also those produced from remodelling simulations on idealized bone structures, are inherently highly pixelated and therefore have a high degree of surface roughness. The purpose of this paper is to demonstrate that this surface roughness need not necessarily have an influence on the predicted properties of the object under examination. To demonstrate this, two-dimensional idealized models of cancellous bone structures were used that were initially depleted and then rebuilt stochastically. A hysteresis effect was observed such that a significant amount of rebuilding beyond the original density was required to regain the initial intact stiffness. To ensure that this effect was not an artefact of the high degree of surface roughness of the rebuilt structures, a two-stage smoothing procedure was applied to assess if this had any effect on the stiffness of the structures. The superpixelation of the structures appeared to have a more profound effect than the smoothing procedures, although the smoothed structures still had stiffness and density values similar to those of the original structures, with a hysteresis effect still evident. This proves that the pixelization of the structures does not have a significant effect on the predicted mechanical properties of the structures. This work has important implications for other models that exhibit a high degree of surface roughness.
Artifacts↗
Structural features of N-glycans linked to royal jelly glycoproteins: structures of high-mannose type, hybrid type, and biantennary type glycans.
The structures of N-glycans of total glycoproteins in royal jelly have been explored to clarify whether antigenic N-glycans occur in the famous health food. The structural feature of N-glycans linked to glycoproteins in royal jelly was first characterized by immunoblotting with an antiserum against plant complex type N-glycan and lectin-blotting with Con A and WGA. For the detail structural analysis of such N-glycans, the pyridylaminated (PA-) N-glycans were prepared from hydrazinolysates of total glycoproteins in royal jelly and each PA-sugar chain was purified by reverse-phase HPLC and size-fractionation HPLC. Each structure of the PA-sugar chains purified was identified by the combination of two-dimensional PA-sugar chain mapping, ESI-MS and MS/MS analyses, sequential exoglycosidase digestions, and 500 MHz 1H-NMR spectrometry. The immunoblotting and lectinblotting analyses preliminarily suggested the absence of antigenic N-glycan bearing beta1-2 xylosyl and/or alpha1-3 fucosyl residue(s) and occurrence of beta1-4GlcNAc residue in the insect glycoproteins. The detailed structural analysis of N-glycans of total royal jelly glycoproteins revealed that the antigenic N-glycans do not occur but the typical high mannose-type structure (Man(9 to approximately 4)GlcNAc2) occupies 71.6% of total N-glycan, biantennary-type structures (GlcNAc2Man3 GlcNAc2) 8.4%, and hybrid type structure (GlcNAc1 Man4GlcNAc2) 3.0%. Although the complete structures of the remaining 17% N-glycans; C4, (HexNAc3 Hex3HexNAc2: 3.0%), D2 (HexNAc2Hex5HexNAc2: 4.5%), and D3 (HexNAc3Hex4HexNAc2: 9.5%) are still obscure so far, ESI-MS analysis, exoglycosidase digestions by two kinds of beta-N-acetylglucosaminidase, and WGA blotting suggested that these N-glycans might bear a beta1-4 linkage N-acetylglucosaminyl residue.
Blotting, Western↗
[The study of chromatin and chromosome structure on preparations of interphase nucleus derivatives resulting from nuclear wall removal. IV. Structural heterogeneity of stretched chromatin in membrane-free cell nuclei from human peripheral lymphocytes].
Densely aggregated chromatin of mature human or animal peripheral lymphocytes is inaccessible for structural investigation on preparations of both intact cell and conventionally spread chromatin. Giemsa- and DAPI-positive "free chromatin" structures, in addition to amembraneous nuclei, were isolated from intact lymphocytes gently treated with Triton-X-100. Surface stretching of both these nuclei and structures, shortly fixed in methanol-glacial acetic acid (3:1), revealed three main types of these "free chromatin" structures: dense chromatin structures (DCS), loose chromatin structures (LCS) and nuclear spreads (NS). The share of each nuclear derivative may be shifted by changing either detergent concentration and(or) the time of incubation in detergent solution. Each DSC consists of condensed "residual" nucleus, similar in from and size with an intact lymphocyte nucleus, and involves 1-15 uni- or olygonemic chromatin sprouts of different length. LSC contain heterogeneously loosened spindle-shape or drop-like nuclei, being several times longer and wider than DCS-nuclei, and 1-3 long uni- or olygonemic chromatin tail-pieces and incidentally observed lateral chromatin sprouts. The majority of LCS contain either a chromocenter of different number of end-to-end associated spindle-shape domains of condensed chromatin. The latter reached 2-5 x 1.5 microns being cross-striated or spiral in structure. NS represent spread chromatin fibrillar structures varying from 150 to 500 microns in length and from 1.5 to almost 50 microns in width. NS consist of 0.3-0.4 micron smooth and 0.4-0.8 micron beaded chromatin fibres. Thin fibres produce web-like domains of NS. and thick fibres form olygonemic bundles or end-to-end association of unit chromatin fibres within NS. Some portion of thick unit fibres of NS gave rise to local splitting into two thin fibres with a similar bead patterns. Thick argyrophilic fibers of the nucleolus also displayed a beaded structure and commonly spread hand-in-hand with the basic chromatin fibre aggregations.
Cell Nucleus↗